In all domains of life, the biosynthesis of complex glycoconjugates requires the concerted action of a multitude of glycosyltransferases (GTs), enzymes that catalyse the transfer of a mono- or oligosaccharide from a glycosyl donor, e.g. a sugar-nucleotide, to a suitable acceptor, e.g. a glycan, peptide or lipid. These functions are further described in Weadge, J. T. & Palcic, M. M. Chemistry of glycosyltransferases. Wiley Encyclopedia of Chemical Biology. DOI 10.1002/9780470048672.wecb213, 1-13 (2008); Lairson, L. L., Henrissat, B., Davies, G. J. & Withers, S. G. Glycosyltransferases: structures, functions and mechanisms. Annu. Rev. Biochem. 77, 521-555 (2008); Schuman, B., Alfaro, J. A. & Evans, S. V. Glycosyltransferase structure and function. Top. Curr. Chem. 272, 217-257 (2008); Breton, C., Snajdrova, L., Jeanneau, C., Koca, J. & Imberty, A. Structures and mechanisms of glycosyltransferases. Glycobiology 16, 29R-37R (2006).
GTs play a key role in many fundamental biological processes underpinning human health and disease, such as cell signalling, cellular adhesion, carcinogenesis, and cell wall biosynthesis in humanpathogens. This is further described in Marth, J. D. & Grewal, P. K. Mammalian glycosylation in immunity. Nat. Rev. Immunol. 8, 874-887 (2008); Rexach, J. E., Clark, P. M. & Hsieh-Wilson, L. C. Chemical approaches to understanding O-GlcNAc glycosylation in the brain. Nat. Chem. Biol. 4, 97-106 (2008); Dube, D. H. & Bertozzi, C. R. Glycans in cancer and inflammation: potential for therapeutics and diagnostics. Nat. Rev. Drug. Discov. 4, 477-488 (2005); Berg, S., Kaur, D., Jackson, M. & Brennan, P. J. The glycosyltransferases of Mycobacterium tuberculosis—roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates. Glycobiology 17, 35R-56R (2007).
The development of small molecular glycosyltransferase inhibitors is therefore of considerable scientific interest in chemical glycobiology and drug discovery. Thus, GT inhibitors are sought after as molecular tools for the interrogation of glycosylation pathways, for mechanistic studies on carbohydrate-active enzymes, and as lead compounds in several important therapeutic areas, including infectious diseases, inflammation and cancer. This is further described in Qian, X. & Palcic, M. M. Glycosyltransferase Inhibitors. In: B. Ernst, G. Hart, P. Sinaÿ (Eds.) Carbohydrates in Chemistry & Biology, p 293-328 (Wiley-VCH, Weinheim, 2000).
A number of methods have been developed to study glycosyltransferases (GTs), as isolated enzymes and in living organisms. These methods allow the monitoring of GT activity during and after enzyme purification and can be used for studies of enzyme mechanisms, inhibition measurements, high throughput screening (HTS) and applications in biocatalysis. Enzymatic bioassays can be designed as either a functional or a binding assay. Functional assays provide information, qualitatively and quantitatively, about the progress of an enzymatically catalysed reaction and about the influence of a chemical of interest on said enzymatic reaction. Thus, the biological activity of a molecule towards an enzyme can be determined, e.g. whether said molecule behaves as an inhibitor or a substrate. Functional bioassays for GTs are most commonly based on monitoring either the depletion of the substrates (i.e. sugar-nucleotide and acceptor) or the formation of the products (i.e. nucleoside diphosphate or glycosylated acceptor).
Ideally, GT functional assays are carried out in real time with saturated substrate concentrations. However, this is often difficult to achieve practically because of the elevated cost and limited availability of many GT substrates. Binding bioassays, on the other hand, do not necessarily require both enzymatic substrates since they are exclusively designed to quantify the binding affinity of a molecule for an enzyme, and do not rely on an enzymatic reaction. Since they can be developed as HTS assays, binding affinity bioassays are especially useful in medicinal chemistry projects to determine the binding activity of small molecular inhibitors.
Due to the complexities of assaying GTs, an extensive range of methods have been employed for the development of both functional and binding assays (Palcic, M. M.; Sujino, K. Trends Glycosci. Glycotechnol. 2001, 13, 361.) Thus, methods based on different principles of detection such as radiochemistry, chromatography, immunology and spectrophotometry have been designed. Functional assays include chromatographic, spectrophotometric and radiochemical assays. The use of chromatographic methods in functional assays is further described in Taniguchi, N.; Nishikawa, A.; Fujii, S.; Gu, J. Methods Enzymol. 1989, 179, 397. Examples of multi-enzyme assay methods using mammalian glycosyltransferases can be found in Gosselin, S.; Alhussaini, M.; Streiff, M. B.; Takabayashi, K.; Palcic, M. M. Anal. Biochem. 1994, 220, 92. Such methods were later adapted for use in microplates allowing high-throughput inhibitor screening. However, these assays often require large quantities of enzymes, limiting their application to highly abundant or cloned enzyme sources.
Radiochemical assays have also been used as functional assays for GTs since they are highly sensitive and enable the detection of low levels of enzymes (Palcic, M. M.; Pierce, M.; Hindsgaul, O. Methods Enzymol. 1994, 247, 215). Typically, the non-continuous assay involves the incubation of the enzyme with radiolabelled sugar-nucleotide and acceptor. After quenching the reaction, several methods exist for the separation of the unreacted radiolabelled donor from the radiolabelled glycosylated product. These include electrophoresis, ion-exchange chromatography, TLC and size exclusion chromatography for glycoproteins. More recently, von Ahsen and coworkers engineered radiochemical assays with suitable conditions for the high throughput screening of drug-like glycosidic acceptor inhibitors. (Von Ahsen, O.; Voigtmann, U.; Klotz, M.; Nifantiev, N.; Schottelius, A.; Ernst, A.; Müller-Tiemann, B; Parczyk, K. Anal. Biochem. 2008, 372, 96.) Their screening of nearly 800,000 compounds enabled the identification of 233 hits, mostly specific to Fucosyltransferase VII, a promising drug target for the treatment of inflammatory skin diseases. The main limitations of this radiochemical assay include hazards associated with the use and disposal of radioactive material and its lack of versatility, since it was exclusively designed for FucTVII.
Other methods available for functional GT assays include Enzyme-Linked Immunosorbant Assays (ELISA), an example of which is described in Verdon, B.; Berger, E. G.; Salchli, S.; Goldhirsch, A.; Gerber, A. Clin. Chem. 1983, 29, 1928. With highly specific antibodies or lectins, immunological assay methods have the advantage of identifying reaction products and being suitable for high throughput screening. Palcic and co-workers also developed a procedure analogous to the ELISA called the ELFIA (Enzyme-Linked Immuno-Fluorescent Assay). In this procedure, originally developed for assaying blood group A and B transferases, BSA-conjugates are coated onto nitrocellulose membranes rather than microplates. Advantageously, this provides a much faster assay than the ELISA technology (Keshvara, L. M.; Gosselin, S.; Palcic, M. M. Glycobiology 1993, 3, 416). Immunological assays based on fluorescence such as “Transcreener Assays” commercialised by BellBrook Laboratories are also available for high throughput GT inhibitor evaluation. Immunological assays are, however, unsuitable for detailed kinetic or mechanistic studies since the acceptor substrate can only be immobilised in low concentrations. Moreover, the availability of antibody or acceptor conjugate can also be a limitation especially for HTS evaluation of large libraries of inhibitors. Many other methods were designed for quantitative GT assays in both isolated enzymes and cells. One of the most recent assays relies on pH measurements and was first reported by Deng and Chen (Deng, C.; Chem, R. R. Anal. Biochem. 2004, 330, 219.) The pH-based assay relies on the detecting the absorbance change of a pH indicator, phenol red, in response to the proton release that accompanies the galactose transfer. Advantageously, the pH-based assay does not require any expensive specialised equipment or labelled substrate, and therefore was successfully applied by Palcic and Persson to automated HTS with mutated GTB enzymes.
Carbohydrate microarrays, often called “lab-on-a-chip”, were also designed for GT activity and the analysis of glycan-protein or glycan-cell interactions as well as for the detection of pathogens (see Nagahori, N.; Niikura, K.; Sadamoto, R.; Taniguchi, M.; Yamagishi, A.; Monde, and K.; Nishimura, S. I. Adv. Synth. Catal. 2003, 345, 729. 46) Park, S.; Shin, I. Org. Lett. 2007, 9, 1675.)
A label-free, real-time glycosyltransferase assay based on exogenic fluorophores such as 8-anilino-1-naphtalenesulfonate (ANS) or artificial zinc-chelated chemosensors has also been developed (see Mizyed, S.; Oddone, A.; Byczynski, B.; Hugues, D. W.; Berti, P. J. Biochemistry 2005, 44, 4011 and Wongkongkatep, J.; Miyahara, Y.; Ojida, A.; Hamachi, I. Angew. Chem. 2006, 118, 681). Attractively, these continuous assays are not limited to specific acceptors, donors or enzymes since their principle of detection only requires cleavage of the donor anomeric linkage. On the other hand, this specific mode of detection makes them unsuitable to assay enzymes other than GTs, and provides only indirect information about the GT reaction, from the formation of the secondary reaction product.
GT ligand-displacement assays, or binding assays, based on fluorescein-labelled sugar-nucleotides have previously been used successfully for the HTS of two GlcNAc transferases, MurG and OGT (see Helm, J. S, Hu, Y., Chen, L., Gross, B. & Walker, S. Identification of Active-Site Inhibitors of MurG Using a Generalizable, High-Throughput Glycosyltransferase Screen. J. Am. Chem. Soc. 125, 11168-11169 (2003). and Gross, B. J., Kraybill, B. C. & Walker, S. Discovery of O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 127, 14588-14589 (2005)). However, for each of these two enzymes an individual, tailor-made fluorophore had to be developed. This limited applicability is a significant drawback, especially as the preparation of each fluorophore required multi-step synthesis.
The present invention aims to provide an alternative to the prior art methods, and may overcome or mitigate at least one problem associated with one or more of the prior art methods, even if not expressly mentioned herein.